Pichia kudriavzevii with intestinal health protection function and application thereof

Abstract

The present invention discloses a Pichia kudriavzevii yeast having the function of protecting intestinal health and its application, and belongs to the field of microbial technology. The Pichia kudriavzevii FZ12 has been deposited in the China Center for Type Culture Collection on January 4, 2023, with the deposit number CCTCCM2023022. The present invention finds that the strain has strong tolerance to temperature, pH, bile salts and artificial gastric juice, and its bacteria and metabolites have strong DPPH free radical scavenging ability. Further animal experiments have found that the strain can significantly improve the growth performance of weaned piglets, reduce diarrhea rate, enhance piglets' immune response ability, promote intestinal development, regulate intestinal microbial balance and increase intestinal short-chain fatty acid concentration, and has the function of protecting intestinal health.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Pichia kudriavzevii yeast having the function of protecting intestinal health and an application thereof. Background Art

[0002] Intestinal health plays a decisive role in host health. The intestine is not only the main place for digestion and absorption of nutrients, but also an important immune barrier of the body. Its health level determines the health of the animal. There are tens of thousands of intestinal microbial flora distributed in the intestine. The stable state of intestinal microorganisms is an important manifestation of the health of the body. The integrity of the intestinal structure and the normal functioning of the intestinal microorganisms are also closely related to intestinal microorganisms. Weaning is an important process in the life cycle of animals. Weaning can lead to a series of weaning stress syndromes, including intestinal flora disorders, villous atrophy, inflammation, and oxidative stress, often accompanied by severe intestinal infections, which further cause diarrhea. Antibiotics are considered to be an effective means of treating diarrhea, but with the emergence of drug-resistant strains, this has caused serious economic and public health problems. Therefore, effective antibiotic alternatives are urgently needed.

[0003] Postbiotics are defined as "inanimate microorganisms and / or their components that are beneficial to the health of the host". Postbiotics include any substances produced by microbial metabolism that directly or indirectly have a beneficial effect on the host. Postbiotics do not contain live bacteria, but have similar functions to probiotics, and have the functions of regulating intestinal flora, maintaining intestinal barrier stability, regulating immune responses, regulating systemic metabolic responses, and other functions to protect intestinal health. Pichia kudriavzevii is widely present in fermented foods, fruits, and wine koji. In addition to producing unique flavor substances, it also has multiple tolerances and unique carbon and nitrogen metabolism. It has great potential in the fields of food, winemaking, and bioenergy. Studies have shown that food-derived P.kudriavzevii has good cadmium and lead binding ability and safety, and its secreted metabolites can inhibit the proliferation of tumor cells. In addition, P.kudriavzevii can be used as a starter and co-culture and is widely used in the preparation of fermented products. There is currently no report on the use of Pichia kudriavzevii fermentation to produce postbiotic products as an antibiotic alternative for preventing and / or treating weaning-induced diarrhea in mammals and promoting host health. Summary of the invention

[0004] The purpose of the present invention is to provide a Pichia kudriavzewi yeast having the function of protecting intestinal health and its application, so as to solve the problems existing in the above-mentioned prior art. The Pichia kudriavzewi yeast FZ12, its metabolites and bacterial lysates can significantly reduce the incidence of diarrhea in weaned piglets and improve their growth performance, thereby effectively protecting intestinal health.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Pichia kudriavzevii FZ12 having the function of protecting intestinal health, which has been deposited in the China Center for Type Culture Collection on January 4, 2023, with the deposit number of CCTCC M 2023022.

[0007] The present invention also provides the use of the Pichia kudriavzevii FZ12 in preparing a medicine for protecting intestinal health.

[0008] Furthermore, protecting intestinal health includes improving intestinal flora structure and alleviating diarrhea.

[0009] Furthermore, the improvement of the intestinal flora structure includes increasing the abundance of intestinal probiotics and reducing the abundance of pathogenic bacteria.

[0010] Furthermore, protecting intestinal health also includes improving the antioxidant capacity of lactobacilli in the intestine.

[0011] Furthermore, the lactobacillus includes Lactobacillus reuteri and Lactobacillus johnsonii.

[0012] The present invention also provides a medicine for protecting intestinal health, comprising the Pichia kudriavzevii FZ12 and / or its metabolites and bacterial lysates.

[0013] The present invention also provides a feed for alleviating diarrhea, wherein the feed comprises the Pichia kudriavzevii FZ12 and / or its metabolites and bacterial lysates.

[0014] The present invention also provides the use of the Pichia kudriavzevii FZ12 in preparing feed for alleviating diarrhea.

[0015] Furthermore, the pig is a weaned piglet.

[0016] The present invention discloses the following technical effects:

[0017] The present invention screens out a strain of Pichia kudriavzewii with strong tolerance (temperature, pH, bile salt and artificial gastric juice) and antioxidant properties. It is found through experimental verification that the Pichia kudriavzewii strain has the effect of alleviating piglet diarrhea caused by weaning, mainly including promoting the development of piglet intestinal villi, increasing the abundance of intestinal indigenous probiotics and reducing the abundance of pathogens, thereby enhancing the immune response ability of piglets, thereby reducing the incidence of diarrhea and improving its production performance. The present invention also finds that the Pichia kudriavzewii strain FZ12 can significantly enhance the growth and biofilm formation of Lactobacillus reuteri and Lactobacillus johnsonii, and can enhance the survival ability of Lactobacillus under oxidative stress. Therefore, the strain can effectively regulate the intestinal flora and enrich intestinal probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 is the phylogenetic tree of Pichia kudriavzevii strain FZ12;

[0020] Figure 2 The temperature (A) and pH (B) tolerance of Pichia kudriavzevii strain FZ12;

[0021] Figure 3 is the tolerance of Pichia kudriavzevii strain FZ12 to artificial gastric juice and bile salts, wherein A is the count of strain FZ12 after being treated with bile salts of different concentrations for 24 hours, B is the survival rate of strain FZ12 after being treated with bile salts of different concentrations for 24 hours, C is the count of strain FZ12 within 4 hours of artificial gastric juice treatment, and D is the survival rate of strain FZ12 within 4 hours of artificial gastric juice treatment;

[0022] Figure 4 is the ability of different strains to scavenge DPPH free radicals, where A is the scavenging rate of filtered supernatants of different strains to DPPH free radicals, and B is the scavenging rate of bacteria of different strains to DPPH free radicals;

[0023] Figure 5 Growth performance (A) and diarrhea condition (B) of weaned piglets in each group;

[0024] Figure 6 Microscopic observation of pathological sections of small intestine of weaned piglets in each group (A), villus height (B), crypt depth (B) and the ratio of villus height to crypt depth (D);

[0025] Figure 7 The goblet cells in each section of the small intestine of weaned piglets in each group, where A is the distribution (left) and quantity (right) of goblet cells in the duodenum of each group, B is the distribution (left) and quantity (right) of goblet cells in the jejunum of each group, and C is the distribution (left) and quantity (right) of goblet cells in the ileum of each group;

[0026] Figure 8 The expression of inflammatory factors in each section of the small intestine of weaned piglets in each group; AF is the detection concentration of IL-6, IL-8, IL-1β, TNF-α, IL-10, and IL-22;

[0027] Fig. 9 The intestinal flora of weaned piglets in each group, where A is a scatter plot of principal coordinate analysis of bacterial communities based on weighted UniFrac distance, and B is a clustering diagram of intestinal bacterial communities in different groups of piglets;

[0028] Fig.10 is the content of short-chain fatty acids in feces of weaned piglets in each group;

[0029] Fig.11 The effect of FZ12 yeast fermentation supernatant on the growth of different Lactobacillus johnsonii and Lactobacillus reuteri;

[0030] Fig.12 Effects of the supernatant of FZ12 yeast fermentation at different fermentation periods on the biomass of Lactobacillus reuteri FP13 (A) and Lactobacillus johnsonii FP47 (B);

[0031] Fig.13 Effects of different addition amounts of FZ12 yeast fermentation supernatant on the biofilm formation ability of Lactobacillus reuteri FP13 (A) and Lactobacillus johnsonii FP47 (B);

[0032] Fig.14 Figure 3 is the effect of FZ12 fermentation supernatant on the antioxidant capacity of lactobacillus, wherein A is the tolerance of Lactobacillus reuteri FP13 to different concentrations of H2O2, B is the tolerance of Lactobacillus johnsonii FP47 to different concentrations of H2O2, C is the effect of FZ12 fermentation supernatant on the antioxidant capacity of Lactobacillus reuteri FP13, and D is the effect of FZ12 fermentation supernatant on the antioxidant capacity of Lactobacillus johnsonii FP47. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The culture medium used in the embodiment of the present invention is as follows:

[0039] YPD medium: 1wt% yeast powder, 2wt% peptone, 2wt% glucose, pH 7.0. Used for Pichia pastoris culture, culture conditions 37°C, 180r / min.

[0040] MRS medium: peptone 10g / L, yeast extract 5g / L, beef extract powder 10g / L, glucose 5g / L, sodium acetate 5g / L, diammonium citrate 2g / L, Tween 80 1g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.05g / L, pH adjusted to 6.8. Used for lactobacillus culture, culture conditions 37℃, stand still.

[0041] CDM medium: glucose 15g / L, arginine 0.72g / L, asparagine 0.5g / L, histidine 0.17g / L, isoleucine 0.24g / L, leucine 1g / L, methionine 0.125g / L, tryptophan 0.3g / L, valine 0.7g / L, biotin 0.006g / L, calcium pantothenate 0.0012g / L, folic acid 0.00056g / L, inositol 0.002g / L, niacin 0.0009g / L, p-aminobenzoic acid 0.000056g / L, pyridoxine hydrochloride 0.0048g / L, riboflavin 0.0009g / L, thiamine hydrochloride 0.00056g / L, ferrous chloride heptahydrate 0.005g / L, Dipotassium hydrogen phosphate 6.48g / L, potassium dihydrogen phosphate 3.12g / L, magnesium chloride 0.3864g / L, sodium chloride 3g / L, zinc sulfate 0.005g / L, potassium sulfate 0.023g / L, boric acid 0.00075g / L, calcium chloride 0.03g / L, cobalt chloride hexahydrate 0.00019g / L, copper sulfate 0.00012g / L, potassium iodide 0.00011g / L, manganese sulfate monohydrate 0.00034g / L, ammonium molybdate tetrahydrate 0.00019g / L, diammonium hydrogen citrate 1.69g / L, citric acid monohydrate 0.003g / L, reduced glutathione 0.015g / L, pH adjusted to 7.0, sterilized by filtration with a 0.22μm sterile filter membrane.

[0042] The weaned piglets involved in the embodiments of the present invention are from a farmer's pig farm in Mianyang, Sichuan. The breed is a Durbin × Chang × Da Sanyuan hybrid pig. The age at weaning is 28 days, and they can eat and drink freely. Animal ethics has been approved by the Ethics Committee of the Animal Center of Huazhong Agricultural University, approval number HZAUSW-2019-011.

[0043] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention can be obtained from common commercial sources.

[0044] Example 1 Isolation, identification and preservation of strains

[0045] 1. Strain Screening

[0046] The liquor lees from Wuliangye Group were used as the separation sample. 10 g of the sample was taken into 90 mL of sterile water and shaken on a shaker for 30 min. The sample was diluted in sequence to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6dilution; 100 μL of gradient dilutions of different gradients were respectively applied on YPD solid medium and cultured at 37°C for 12 h. Typical colonies on the dilution plate were streaked on YPD solid medium and cultured at 37°C for 12 h to obtain purified colonies; purified colonies were inoculated into YPD liquid medium and cultured at 37°C for 12 h to obtain strain FZ12 and seed solution.

[0047] 2. Strain Identification

[0048] The genome of FZ12 was extracted, and the ITS, 18S rDNA and 26S rDNA sequences of FZ12 were amplified and sequenced (completed by Shanghai Sangon Biotechnology Co., Ltd.). The sequenced sequences were compared and analyzed in the NCBI database, and a phylogenetic tree was constructed based on the 26S rDNA sequence ( Figure 1 ), Figure 1 The results showed that the strains were all Pichia kudriavzevii, and were named Pichia kudriavzevii FZ12.

[0049] 3. Strain preservation

[0050] Pichia pastoris FZ12 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on January 4, 2023, with the deposit number CCTCC M 2023022, and is referred to as Pichia pastoris FZ12.

[0051] Example 2 Temperature and pH tolerance determination of Pichia kudriavzevii FZ12

[0052] Prepare YPD liquid medium with 20g / L glucose, add 200mL medium to a 500mL Erlenmeyer flask, inoculate with 1% FZ12 strain seed solution, and culture in a shaker at 37℃, 42℃, and 50℃, respectively, with a rotation speed of 200r / min. Measure OD every 2h. 600 , until the OD 600 The values ​​were basically unchanged, and 3 replicates were performed at each temperature.

[0053] Prepare YPD liquid medium with 20g / L glucose, adjust the pH to 1, 3, 5, 7, 9, 11 with concentrated HCl, 3 replicates each, add 200mL medium to each 500mL Erlenmeyer flask, inoculate with 1% seed solution, set the temperature to 37℃, and rotate at 200rpm. Measure OD every 2h 600 , until the OD 600 The values ​​were basically unchanged, and 3 replicates were performed at each temperature.

[0054] The growth status of strain FZ12 under different temperatures and pH conditions is as follows Figure 2 As shown in the figure, the FZ12 strain grows best and fastest at 37°C, and still grows well when the temperature reaches 42°C. However, when the temperature rises to 45°C, FZ12 hardly grows. In addition, the FZ12 strain also has a strong tolerance to pH. When the pH is 1, the strain stops growing. However, when the pH is between 3-11, the strain grows well, and the optimal pH is 7.0. Therefore, the strain FZ12 is a strain that is resistant to high temperatures, acids and alkalis.

[0055] Example 3 Determination of tolerance to bile salts and artificial gastric juice of Pichia kudriavzevii FZ12

[0056] Prepare YPD medium with porcine bile salt concentrations of 0.5%, 1%, 1.5% and 2% respectively. After activating the strain, take 1 mL of the culture solution and inoculate it into 9 mL of YPD medium with each bile salt concentration. After static culture at 37°C for 24 hours, dilute and spread the culture medium for counting, with 3 parallels for each concentration.

[0057] Adjust the pH of the prepared artificial gastric juice to 2.5, sterilize and set aside. After activating the strain, take 1 mL of culture medium (1-2×10 9 CFU / mL), inoculated into 9 mL of artificial gastric juice, cultured at 37°C, and sampled and diluted for smear counting every 1 h.

[0058] The survival rate formula is calculated as follows:

[0059]

[0060] Where X0——the number of yeast at 0h, the unit is CFU / mL;

[0061] X n ——The number of yeast cells after 6 hours or 24 hours, in CFU / mL.

[0062] The tolerance of strain FZ12 to artificial gastric juice and bile salts is as follows Figure 3 As shown by Figure 3 From A and B, we can see that the FZ12 strain has a strong tolerance to bile salts. As the bile salt concentration increases, the number of FZ12 bacteria decreases to a certain extent compared with the initial level, but the survival rate remains between 87% and 89%. Normally, the bile salt concentration tolerance of probiotics is generally 0.3%-0.5%, but when the bile salt concentration reaches 2%, the survival rate of FZ12 still reaches 87%, which shows that the FZ12 strain has a strong tolerance to bile salts.

[0063] Depend on Figure 3 As shown in C and D, the FZ12 strain has a strong tolerance to artificial gastric juice. As the treatment time increases, the bacterial count increases from the initial 4×109 The CFU / mL was reduced to 5.6×10 8 CFU / mL, although the number of viable bacteria decreased to a certain extent, the survival rate was always above 80% after 4 h of treatment. However, when the treatment time was 1 h, the survival rate reached 90%, indicating that FZ12 had a strong tolerance to artificial gastric juice and had a strong intestinal survival ability.

[0064] Example 4 DPPH free radical scavenging activity of Pichia kudriavzevii FZ12 strain

[0065] First, prepare the resuspension and fermentation supernatant of yeast and control strains, and use VC as a positive control. Activate FZ12, Saccharomyces cerevisiae Sc, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri ATCC6475, pick single colonies of the above strains, use YPD medium for yeast and MRS medium for lactobacillus, culture at 37°C for 24h, and transfer twice as seed liquid. Prepare YPD and MRS medium, inoculate the above strains into cell bottles containing 30mL YPD or MRS medium, respectively, and place FZ12 and Sc in a 37°C and 30°C shaker, respectively, and culture at 200r / min for 48h. Lactobacillus is cultured statically in a 37°C incubator for 24h. Then centrifuge to collect the cells, resuspend with an equal volume of PBS to obtain a resuspension of the cells. At the same time, filter the supernatant with a 0.22μm sterile filter membrane to sterilize and obtain a cell-free fermentation supernatant.

[0066] DPPH was completely dissolved in ethanol to a concentration of 200mmol / L. 1.0mL of the prepared DPPH solution was taken and mixed with 0.8mL of yeast resuspension and yeast fermentation supernatant, respectively. The mixture was placed at room temperature for 30min, centrifuged at 2000×g for 2min, and the absorbance of the reaction solution was measured at 517nm. PBS was used as a blank control, and the free radical scavenging ability was calculated as follows:

[0067]

[0068] Where: A x ——Absorbance after adding DPPH and yeast or extract;

[0069] A0——Absorbance after adding DPPH and PBS.

[0070] Depend on Figure 4 It can be seen that the FZ12 strain has a strong DPPH free radical scavenging ability. The DPPH scavenging ability of the fermentation supernatant of the strain FZ12 reached 82.46%, which was significantly better than the fermentation supernatant of Saccharomyces cerevisiae (74.01%), and there was no significant difference in DPPH scavenging ability with LGG, ATCC6475 strains and VC ( Figure 4However, in terms of the DPPH scavenging ability of the bacteria, the DPPH scavenging ability of FZ12 reached 70.27%, which was significantly higher than that of other strains ( Figure 4 B). Therefore, for the FZ12 strain, both the fermentation supernatant and the bacterial cells have strong DPPH scavenging ability, and its potential antioxidant capacity also has obvious advantages.

[0071] Example 5. Effect of Pichia kudriavzevii FZ12 culture on weaned piglets

[0072] 1. Grouping and feeding of weaned piglets

[0073] 72 healthy weaned piglets with similar body weight were selected and randomly divided into 3 groups. The piglets in each group were raised in 3 pens (8 piglets in each pen). The three experimental groups were the non-antibiotic group (FN), the FA group (FA), and the FZ12 culture group (FYC). The piglets in the non-antibiotic group were fed with a basic feed, the piglets in the FA group were fed with a basic feed supplemented with antibiotics (enramycin, quinocetone, and oxytetracycline), and the piglets in the FZ12 culture group were fed with a basic feed supplemented with 0.5% of the solid-state fermentation lysate of Pichia kudriavzevii FZ12. The feed intake and diarrhea were recorded daily. The feces of the piglets were collected in the early morning of the 0th, 3rd, 6th, and 12th days after early weaning (piglets were weaned at 28 days of age, and the 0th day of early weaning corresponded to the first day of group feeding).

[0074] The solid-state fermentation lysate of Pichia azewieri FZ12 is produced by solid-state fermentation of Pichia azewieri: using bran as a substrate, solid-state fermentation is carried out at 37°C for 72 hours, autolysis is carried out at 45°C under natural pH conditions for 48 hours, and the fermentation lysate is dried and crushed at 50°C to obtain the fermentation lysate.

[0075] 2. Effects of FZ12 culture on diarrhea and growth of weaned piglets

[0076] The average daily feed intake, daily weight gain and diarrhea results of weaned piglets in each group are as follows: Figure 5 And as shown in Table 1.

[0077] Table 1 Statistics of growth performance of weaned piglets

[0078]

[0079] Note: Significance ( a , b ,p<0.05).

[0080] The addition of FZ12 culture significantly increased the average daily feed intake and daily weight gain. As an important indicator of growth performance, the feed conversion rate of the FZ12 culture group was significantly higher than that of the antibiotic-free group, and the feed conversion rate increased by 20.06%, while there was no significant difference with the antibiotic group; the use of FZ12 culture and antibiotics significantly reduced the incidence of diarrhea, from 19.79±1.47% (antibiotic-free group) to 8.33±0.85% (antibiotic group) and 13.54±0.85% (FZ12 culture group). Compared with the antibiotic-free group, the diarrhea rate in the FZ12 culture group was reduced by 31.58%. Similarly, the diarrhea index was significantly reduced using FZ12 culture, from 0.54±0.05 in the antibiotic-free group to 0.33±0.02 (FZ12 culture group), and the diarrhea index was reduced by 38.89%.

[0081] 3. Effects of FZ12 culture on the intestine of weaned piglets

[0082] On the 12th day of feeding, one piglet was randomly selected from each pen for dissection and blood collection, and duodenum, jejunum, and ileum tissue samples were taken for subsequent experimental analysis.

[0083] 3.1 Effects on the number of intestinal and goblet cells in weaned piglets

[0084] Take appropriate samples and fix them with paraformaldehyde for pathological section analysis. Figure 6 As shown. Another appropriate sample was fixed with paraformaldehyde and then stained with PAS for the analysis of the number of intestinal goblet cells. Figure 7 shown.

[0085] like Figure 6 The BD showed that the addition of Pichia kudriavzevii culture significantly increased the height of the villi in each segment of the small intestine and decreased the crypt depth in the jejunum compared with the other two groups. In both the jejunum and ileum, the FZ12 culture group significantly increased the ratio of villus height to crypt depth.

[0086] like Figure 7 It can be seen that compared with the non-antibiotic group, FZ12 culture significantly promoted the number of goblet cells on the villi of the duodenum (7A), jejunum (7B), and ileum (7C).

[0087] 3.2 Effects on intestinal inflammatory factors in weaned piglets

[0088] Take appropriate samples and grind and lyse the intestinal samples using tissue lysis buffer (Biosharp, 70010050). The Jiangsu enzyme-immune porcine interleukin-10 enzyme-linked immunosorbent assay kit (MM-0425O1) was used to detect the content of interleukin-10 (IL-10) in small intestinal tissue. The Jiangsu enzyme-immune porcine interleukin-6 enzyme-linked immunosorbent assay kit (MM-0418O1) was used to detect the content of interleukin-6 (IL-6) in small intestinal tissue. The Jiangsu enzyme-immune porcine interleukin-8 enzyme-linked immunosorbent assay kit (MM-37057O1) was used to detect the content of interleukin-8 (IL-8) in small intestinal tissue. The Jiangsu enzyme-immune porcine interleukin-22 enzyme-linked immunosorbent assay kit (MM-1231O1) was used to detect the content of interleukin-22 (IL-22) in small intestinal tissue. Jiangsu Enzyme Immunity Porcine Interleukin-1β ELISA Kit (MM-0422O1) was used to detect the content of interleukin-1β (IL-1β) in small intestinal tissue. Jiangsu Enzyme Immunity Porcine Interferon-γ ELISA Kit (MM-0412O1) was used to detect the content of interferon-γ (IFN-γ) in small intestinal tissue. Jiangsu Enzyme Immunity Porcine Tumor Necrosis Factor α ELISA Kit (MM-0383O1) was used to detect the content of tumor necrosis factor-α (TNF-α) in small intestinal tissue. Specific operations were carried out according to the instructions of the kit.

[0089] The results are as follows Figure 8 As shown, the addition of FZ12 yeast culture significantly reduced the levels of pro-inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α in small intestinal tissue, and increased the levels of anti-inflammatory cytokines IL-10 and IL-22.

[0090] 4. Effects of FZ12 culture on intestinal flora structure and short-chain fatty acid content in feces of weaned piglets

[0091] A fecal sample of one piglet was randomly selected from each pen and divided into three portions. One portion was preserved in glycerol for strain screening, and the other two portions were used for bacterial 16S rDNA amplification sequencing analysis and short-chain fatty acid content analysis. After the samples were collected, they were immediately placed in dry ice and stored at -80°C.

[0092] The intestinal flora structure of weaned piglets and the detection of short-chain fatty acids in feces are as follows: lactic acid is detected by high performance liquid chromatography, and acetic acid, propionic acid and butyric acid are detected by gas chromatography:

[0093] (1) Plotting of standard curve. The concentration of lactic acid standard is 80 g / L, which is diluted to 40 g / L, 20 g / L, 10 g / L, 5 g / L, 2.5 g / L, and 1.25 g / L. The concentration of acetic acid standard is 80 g / L, which is diluted to 40 g / L, 20 g / L, 10 g / L, 5 g / L, 2.5 g / L, and 1.25 g / L. The concentration of propionic acid standard is 20 g / L, which is diluted to 10 g / L, 2.5 g / L, 1.25 g / L, 0.625 g / L, and 0.3125 g / L. The concentration of butyric acid standard is 10 g / L, which is diluted to 5 g / L, 2.5 g / L, 1.25 g / L, 1 g / L, 0.625 g / L, 0.3125 g / L, and 0.1 g / L. When gas phase detection is used, 2-ethylbutyric acid is selected as the internal standard. A standard curve is drawn based on the peak area and the corresponding standard substance concentration.

[0094] (2) Take the piglet feces out of the -80℃ freezer and place them in liquid nitrogen.

[0095] (3) Prepare a centrifuge tube, add 1 mL of sterile water and a small steel ball, add 100 mg of feces and mark it, place in an ice bath for 5 min, and homogenize for 50 s at 40 Hz, 1-2 times.

[0096] (4) After vortexing, the sample was placed on ice for 5 min and then centrifuged at 3000 g for 5 min at 4 °C. The supernatant was aspirated.

[0097] (5) Adjust the pH of the supernatant to 2-3 with 5 mol / L HCl and leave at room temperature for 10 min.

[0098] (6) Centrifuge at 12,000 g for 10 min at 4°C, remove 500-600 μL of supernatant, add an equal volume of internal standard for gas phase detection, pass through the membrane into the injection bottle, and then load on the detector.

[0099] (7) Gas chromatography conditions: the chromatographic column was an Agilent DB-FFAP column (30 m × 0.32 mm × 0.25 μm); the carrier gas was high-purity nitrogen (99.99%) with a flow rate of 1.5 ml / min; the auxiliary gases were hydrogen and air, with a hydrogen flow rate of 30 mL / min and an air flow rate of 400 mL / min; the detector FID temperature was 270 °C; no split; the injection volume was 2 μL; the injection port was 250 °C; the programmed temperature was: initial temperature: 80 °C, equilibrated for 5 min, heated to 220 °C at a rate of 10 °C / min, and run for 22 min.

[0100] (8) HPLC conditions: Bio-Rad HPX-87H ion-exclusion column, mobile phase 5 mM H2SO4, column temperature 40°C, UV detector (210 nm), flow rate 0.6 mL / min, injection volume 20 μL.

[0101] The results of the test on the intestinal flora structure of weaned piglets are as follows Fig. 9 The addition of FZ12 yeast culture significantly changed the intestinal flora structure of weaned piglets and significantly enriched 22 beneficial intestinal bacteria, among which the abundance of Lactobacillaceae changed the most. It also significantly reduced the relative abundance of Spirochaeta, Shigella and Campylobacter, which cause diarrhea in weaned piglets.

[0102] The results of the test on the content of short-chain fatty acids in the feces of weaned piglets are as follows: Fig.10 , on days 0 and 3 after weaning, there were no significant differences in the concentrations of propionic acid, acetic acid, and butyric acid among the groups. On day 6 after weaning, there were no significant differences in the concentrations of propionic acid, acetic acid, and butyric acid. The concentrations of acetic acid and butyric acid in the FZ12 culture group were significantly higher than those in the other groups. In addition, the concentrations of short-chain fatty acids in the FZ12 culture group on day 12 after weaning were significantly higher than those in the other groups.

[0103] Example 6 Effect of Pichia kudriavzevii FZ12 fermentation supernatant on Lactobacillus

[0104] 1. Effect on the growth of Lactobacillus

[0105] Take a ring of glycerol tubes to preserve Pichia kudriavzevii FZ12, streak it on a solid YPD plate, and culture it at 37℃ for 24h; pick a single colony in 10mL YPD liquid culture medium, culture it at 37℃ for 24h, and transfer it twice as seed liquid. Prepare 500mL YPD culture medium, divide it into 5 500mL sterile triangular flasks, each containing 100mL culture medium, inoculate the seed liquid into the above triangular flasks at a 1% (v / v) inoculation amount, place it in a 37℃ shaker, and culture it at 200r / min. Collect the fermentation supernatant at 8h, 24h, 48h, 72h, and 96h, filter it with a 0.22μm sterile filter membrane for sterilization, and store it at -80℃ for use.

[0106] Activate the Lactobacillus johnsonii and Lactobacillus reuteri strains isolated from feces, streak the strains on a solid MRS plate, culture at 37°C for 24 hours, pick a single colony in 10 mL MRS liquid culture medium, culture at 37°C for 24 hours, and transfer twice as seed liquid. Inoculate the seed liquid at a 2% inoculation rate into CDM medium containing 30% of 0h, 8h, 24h, 48h, 72h, and 96h FZ12 fermentation supernatant, place the centrifuge tube in a 37°C shaker, culture at 200r / min for 12 hours, and set 4 replicates for each group. Determine the OD of each group 600 , statistical data.

[0107] like Fig.11 As shown in the figure, three strains of Lactobacillus reuteri and three strains of Lactobacillus johnsonii were randomly selected for the experiment. The FZ12 yeast fermentation supernatant had different promoting effects on different strains. The addition of FZ12 yeast fermentation supernatant increased the OD of Lactobacillus reuteri. 600 The effect of yeast fermentation supernatant on Lactobacillus johnsonii OD 600 The promotion multiples ranged from 1.5 to 4.5 times, with the best effect on FP47. 600 An increase of 4.23 times.

[0108] Depend on Fig.12 It can be seen that with the increase of fermentation period, the promoting effect on Lactobacillus reuteri increased significantly, and when the 12h fermentation supernatant was added, the growth promoting effect was significantly increased, OD 600 The growth-promoting effect reached the highest level when the supernatant of 72h fermentation was added. 600 For Lactobacillus johnsonii FP47, the growth-promoting effect first decreased and then increased. In the 12-60h stage, the growth-promoting effect was significantly lower than that of the supernatant at 0h of fermentation. When the supernatant at 72h of fermentation was added, the growth-promoting effect increased significantly and reached the highest value, making OD 600 It increased from 0.197 to 0.599, an increase of 2.04 times.

[0109] 2. Effects on Lactobacillus biofilm formation

[0110] Lactobacillus reuteri FP13 and Lactobacillus johnsonii FP47 were selected for further study. The lactobacilli to be tested were cultured at 37°C overnight, and then the OD values ​​of each group were unified. 600=0.5, inoculated in 2mL of MRS medium containing 2%, 4%, 6%, 8%, 10%, 20%, 30% FZ12 yeast 72h fermentation culture medium, and then dispensed into 96-well cell culture plates, 200μL per well, for a total of 8 replicates. Place in an automatic growth curve analyzer, culture at 37°C, 200r / min, for 12h, and monitor growth at the same time. After the culture is completed, remove the culture medium, wash with distilled water 3 times, and wash away the planktonic bacteria. After drying, stain with 0.1% crystal violet for 20min. After staining, rinse the staining solution with distilled water and dry. Elute with 33% acetic acid and detect the absorbance at OD 595nm.

[0111] Depend on Fig.13 It can be seen that the FZ12 yeast fermentation supernatant has a very obvious effect on enhancing the biofilm formation ability of Lactobacillus reuteri. With the increase in the amount of yeast fermentation supernatant added, the amount of biofilm formation of Lactobacillus reuteri increased by 1.82-6.55 times. When 2% of yeast fermentation supernatant was added, the amount of biofilm formation increased by 1.82 times. When 10% of yeast fermentation supernatant was added, the increase reached a maximum of 6.55 times. However, when the addition amount exceeded 10%, the enhancement effect began to decrease. For Lactobacillus johnsonii, yeast fermentation supernatant also has a significant enhancement effect. The addition of different yeast fermentation supernatants increased the biofilm formation ability by 1.29-1.55 times. With the increase in the addition amount, the biofilm yield increased accordingly, reaching the highest when the addition amount reached 8%, with an increase of 1.55 times. When the addition amount was greater than 8%, there was no significant difference in the increase in the biofilm yield.

[0112] 3. Effects on the antioxidant capacity of Lactobacillus

[0113] Lactobacillus reuteri FP13 and Lactobacillus johnsonii FP47 strains were activated, and MRS medium was used as the control group. MRS medium was added with 0mmol / L, 1mmol / L1.5mmol / L, 2mmol / L and 3mmol / L hydrogen peroxide as the experimental group to explore the hydrogen peroxide tolerance of Lactobacillus reuteri and Lactobacillus johnsonii ( Fig.14 A and B), Lactobacillus reuteri is tolerant to 1mmol / L hydrogen peroxide, and when the hydrogen peroxide concentration rises to 1.5mmol / L, the growth of Lactobacillus reuteri is significantly inhibited. However, Lactobacillus johnsonii has poor tolerance to hydrogen peroxide, and 0.5mmol / L hydrogen peroxide has a significant growth inhibitory effect. Subsequently, 2mmol / L was selected as a suitable hydrogen peroxide concentration to be added to MRS, and 30% yeast culture was added at the same time. The lactobacillus seed liquid was inoculated and cultured at 2%, and cultured at 37°C, 200r / min, for 12h, and the OD was detected. 600 nm. The results are as follows Fig.14C and D, when 2mmol / L hydrogen peroxide exists, the growth of Lactobacillus reuteri and Lactobacillus johnsonii is significantly inhibited. After the additional addition of yeast fermentation supernatant, the antioxidants in the yeast fermentation supernatant remove the hydrogen peroxide, and some effective substances also promote the growth of Lactobacillus. Therefore, when the hydrogen peroxide concentration inhibits the growth of Lactobacillus, the yeast fermentation supernatant relieves the inhibitory pressure and significantly promotes the growth of Lactobacillus.

[0114] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Pichia kudriavzevii yeast ( Pichia kudriavzevii ) The use of FZ12 in the preparation of a drug for protecting the intestinal health of weaned piglets, characterized in that: The Pichia pastoris FZ12 was deposited in the China Center for Type Culture Collection on January 4, 2023, with the deposit number CCTCCM2023022; The protection of intestinal health includes improving the intestinal flora structure, villous atrophy, inflammation and oxidative stress and alleviating diarrhea of ​​weaned piglets.

2. The use according to claim 1, characterized in that: The improvement of the intestinal flora structure includes increasing the abundance of intestinal probiotics and reducing the abundance of pathogenic bacteria.

3. The use according to claim 1, characterized in that: The protecting of intestinal health also includes improving the antioxidant capacity of lactobacilli in the intestine.

4. The use according to claim 3, characterized in that: The lactobacilli include Lactobacillus reuteri and Lactobacillus johnsonii.

5. A drug for protecting intestinal health, characterized in that: The method comprises the Pichia kudriavzevii FZ12 and / or its bacterial lysate as described in claim 1.

6. A feed for alleviating diarrhea, characterized in that: The feed comprises the Pichia kudriavzevii FZ12 and / or its bacterial lysate as described in claim 1.

7. Use of the Pichia kudriavzevii FZ12 as claimed in claim 1 in preparing feed for alleviating diarrhea in weaned piglets.

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